The present invention relates to analogs that display selective inhibitory activity against the aspartyl proteases, plasmepsin and cathepsin D.
Resistance to known antimalarial therapies is becoming an increasing problem and new therapies are therefore desperately needed. Upon infecting a host, the malaria parasite avidly consumes the host hemoglobin as its source of nutrients. Plasmepsin I and II are proteases from Plasmodium falciparum that are necessary during the initial stages of hemoglobin hydrolysis and digestion, which occurs in the xcex1-chain, between Phe 33 and Leu 34, then other sites serve as substrates for hydrolysis as well. In culture, inhibition of plasmepsin by a peptidomimetic inhibitor is demonstrated as effective in preventing malarial hemoglobin degradation and in killing the parasite (Francis, S. E., Gluzman, I. Y., Oksman, A., Knickerbocker, A., Mueller, R., Bryant, M. L., Sherman, D. R., Russell, D. G., and Goldberg, D. E. (1994) EMBO J, 13, 306-317). Thus, persons of skill in the art expect that plasmepsin inhibitors will provide effective antimalarial therapy in humans.
Cathepsin D is a human protease in the endosomal-lysosomal pathway, involved in lysosomal biogenesis and protein targeting, and may also be involved in antigen processing and presentation of peptide fragments. The protease therefore displays broad substrate specificity, but prefers hydrophobic residues on either side of the scissile bond.
Cathepsin D has been implicated in a variety of diseases, including connective tissue disease, muscular dystrophy, and breast cancer. Cathepsin D is also believed to be the protease which processes the xcex2-amyloid precursor protein (Dreyer, R. N., Bausch, K. M., Fracasso, P., Hammond, L. J., Wunderlich, D., Wirak, D. O., Davis, G., Brini, C. M., Bucholz, T. M., Konig, G., Kamarck, M. E., and Tamburini, P. P. (1994) Eur. J. Biochem., 224, 265-271 and Ladror, U. S., Synder, S. W., Wang, G. T., Holzman, T. F., and Krafft, G. A. (1994) J. Biol. Chem., 269, 18422-18428), generating the major component of plaques in the brains of Alzheimer""s patients. Consequently, persons of skill in the art expect that inhibitors of cathepsin D will be useful in treating Alzheimer""s disease.
The present invention relates to peptidomimetic (hydroxystatine amides and hydroxyphosphonates) analogs and their inhibitory action against aspartyl proteases. More particularly, the invention relates to the identification of such compounds that display selective inhibitory activity against plasmepsin and cathepsin D. Although statine-containing peptides are known which inhibit aspartyl proteases (Shewale, J. G. ; Takahashi, R., Tang, J., Aspartic Proteinases and Their Inhibitors, Kostka, V., Ed. Walter de Gruyter: Berlin (1986) pp. 101-116; U.S. Ser. No. 08/743,944, filed Nov. 5, 1996 which is hereby incorporated by reference in its entirety), there are only a few selective inhibitors for cathepsin D (Lin, T. -Y.; Williams, H. R., Inhibition of Cathepsin D by Synthetic Oligopeptides, J. Biol. Chem. (1979), 254, 11875-11883; Rich, D. H.; Agarwal, N. S., Inhibition of Cathepsin D by Substrate Analogues Containing Statine and by Analogues of Pepstatin, J. Med. Chem. (1986) 29 (2519-2524)), and for plasmepsin (Silva, A. M. et al., Structure and Inhibition of Plasmepsin II, A Hemoglobin-Degrading Enzyme From Plasmodiumfalciparum, Proceed Natl Acad Sci, 1996, 93, 10034-10039).
The present invention also relates to the solid phase synthesis of such peptidomimetic analogs.
I. Preferred Embodiments
The compounds of the present invention are represented by Formula I: 
wherein:
R1 is chosen from the group consisting of alkyl, xe2x80x94(CH2)n-cycloalkyl, xe2x80x94(CH2CH2)nNHC(O)-alkyl, and arylalkyl, wherein n=1-3;
R2 is H or Ŝ xe2x80x94C(O)-L- wherein Ŝ is a solid support, and -L-, is a linker;
Y is xe2x80x94P(O)(OR3)2 or xe2x80x94CH(OH)C(O)NR4R5, wherein R3 is alkyl, arylalkyl, or haloalkyl; and R4 and R5 are independently chosen from the group consisting of H, alkyl, xe2x80x94(CH2)n-cycloalkyl, xe2x80x94(CH2CH2)nNHC(O)-alkyl, arylalkyl, 
xe2x80x94C(H)(R9)CH2OR10, xe2x80x94C(H)(R11)C(H)(OR10)(R11),-alkyl-NHSO2R11, xe2x80x94C(H)(R9)C(O)NHR10, and xe2x80x94C(H)(R9)C(O)NHC(H)(R9)C(O)NHR10, wherein n=1-3;
R9 is independently selected from the group consisting of alkyl and arylalkyl;
R10 is independently selected from the group consisting of H, alkyl, and arylalkyl,
R11 is independently selected from the group consisting of alkyl and aryl; or, when taken together, R4 and R5 can be 
wherein
R12 and R13 are independently selected from the group consisting of H, halo, and alkoxy; and
Z is xe2x80x94C(O)R6 and xe2x80x94C(O)C(H)(R7)OC(O)NHR8, wherein R6 is alkyl, arylalkyl, aryl, xe2x80x94(CH2)m-cycloalkyl, heteroaryl, or 
wherein m=0-3;
R7 is H, alkyl, arylalkyl, or xe2x80x94(CH2)n-cycloalkyl; and
R8 is alkyl, arylalkyl, or aryl.
Preferred compounds of Formula I are those wherein -L- is of Formula (a) 
wherein the designated meta-position is attached to the xe2x80x94C(O)xe2x80x94and the ortho-methylene attaches to the amide nitrogen of Formula I.
A preferred embodiment of the invention are compounds of Formula I wherein:
Y is xe2x80x94P(O)(OR3)2, wherein R3 is arylalkyl; and 
Another preferred embodiment of the invention are compounds of Formula I wherein:
Y is xe2x80x94C(H)(OH)C(O)NHR5, wherein R5 is 
wherein R9 is independently selected from the group consisting of alkyl and arylalkyl; and 
wherein R8 is alkyl or arylalkyl.
A further preferred embodiment of the invention are compounds of Formula I wherein:
Y is xe2x80x94C(H)(OH)C(O)NHR5, wherein R5 is xe2x80x94C(H)(R11)C(H)(OR10)(R11), wherein
R11 is aryl and R10 is H, wherein each R11 may be the same or different; and 
wherein R8 is alkyl or arylalkyl.
Yet another preferred embodiment of the invention are compounds of Formula I wherein:
Y is xe2x80x94C(H)(OH)C(O)NHR5, wherein R5 is xe2x80x94C(H)(R11)C(H)(OR10)(R11), wherein
R11 is aryl and R10 is H, wherein each R11 may be the same or different; and
Z is R6C(O)xe2x80x94, wherein R6 is alkyl, arylalkyl, xe2x80x94(CH2)m-cycloalkyl, or 
Another aspect of the invention is the use of divinylbenzene-cross-linked, polyethyleneglycol-grafted polystyrene beads optionally functionalized with amino groups (e.g., TentaGel(trademark) S NH2, Rapp Polymere) as the solid supports for constructing compounds of Formula I.
II. Abbreviations and Definitions
The following abbreviations and terms have the indicated meanings throughout:
Ac=Acetyl
BNB=4-bromomethyl-3-nitrobenzoic acid
BOC=t-butyloxycarbonyl
BSA=bovine serum albumin
Bu=butyl
c-=cyclo
DABCYL=4-(4-dimethylaminophenylazo)benzoic acid
DBU=Diazabicyclo[5.4.0]undec-7-ene
DCM=Dichloromethane=methylene chloride=CH2Cl2 
DIC=diisopropylcarbodiimide
DIEA=diisopropylethyl amine
DMAP=4-N,N-dimethylaminopyridine
DMF=N,N-dimethylformamide
DMSO=Dimethyl sulfoxide
DVB=1,4-divinylbenzene
EDANS=5-[(2-aminoethyl)amino]naphthalene-1-sulfonic acid
Et=ethyl
Fmoc=9-fluorenylmethoxycarbonyl
HOAc=acetic acid
HOBt=hydroxybenzotriazole
IBX=iodoxybenzoic acid
LiI=lithium iodide
m-=meta
Me=methyl
NMO=N-methylmorpholine oxide
o-=ortho
PEG=polyethylene glycol
Ph=phenyl
PfP=pentafluorophenol
r.t.=room temperature
sat""d=saturated
s-=secondary
t-=tertiary
TBS=tert-butyldimethylsilyl
TFA=trifluoroacetic acid
THF=tetrahydrofuran
TMS=trimethylsilyl
Tris=tris(hydroxymethyl)aminomethane
UV=ultraviolet light
xe2x80x9cAlkoxyxe2x80x9d means alkoxy groups of from 1 to 8 carbon atoms of a straight, branched, or cyclic configuration and combinations thereof Examples of alkoxy groups include methoxy, ethoxy, propoxy, isopropoxy, cyclopropyloxy, cyclohexyloxy, and the like.
xe2x80x9cAlkylxe2x80x9d is intended to include linear or branched hydrocarbon structures and combinations thereof. xe2x80x9cLower alkylxe2x80x9d means alkyl groups of from 1 to 12 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, s-and t-butyl, pentyl, hexyl, octyl, and the like.
xe2x80x9cArylxe2x80x9d is a 6-membered or 10-membered aromatic ring system where each of the rings is optionally substituted with 1-3 substituents selected from alkyl, halogen, hydroxy, alkoxy, aryloxy, alkoxyethoxy, haloalkyl, phenyl, heteroaryl; and wherein the phenyl is optionally substituted with 1-3 substituents selected from alkyl, halogen or alkoxy. Examples of aryl groups are phenyl, 3,4-dimethoxyphenyl and naphthyl.
xe2x80x9cArylalkylxe2x80x9d means an alkyl containing an aryl ring. For example: benzyl, phenethyl, 4-chlorobenzyl, and the like.
xe2x80x9cAryloxyxe2x80x9d means a phenoxy group where the phenyl ring is optionally substituted with 1 to 2 groups selected from halo, alkoxy, or alkyl.
xe2x80x9cCycloalkylxe2x80x9d includes cyclic hydrocarbon groups of from 3 to 12 carbon atoms. Examples of xe2x80x9ccycloalkylxe2x80x9d groups include c-propyl, c-butyl, c-pentyl, c-hexyl, 2-methylcyclopropyl, norbornyl, adamantyl, and the like.
xe2x80x9cHaloalkylxe2x80x9d means that one or more hydrogen atoms present in an alkyl group are substituted with a halogen atom, except for the methylene hydrogens adjacent to the oxygen atom. For example: 2-chloroethyl, and 2,2,2-trifluoroethyl.
xe2x80x9cHalogenxe2x80x9d includes F, Cl, Br, and I, with F and Cl as the preferred groups.
xe2x80x9cHeteroarylxe2x80x9d means a 5- or 6-membered heteroaromatic ring containing 0-2 heteroatoms selected from O, N, and S; or a bicyclic 9- or 10-membered heteroaromatic ring system containing 0-2 heteroatoms selected from O, N, and S; where the methine H atom may be optionally substituted with alkyl, alkoxy or halogen. The 5- to 10-membered aromatic heterocyclic rings include imidazole, pyridine, indole, thiophene, benzopyranone, thiazole, furan, benzimidazole, quinoline, isoquinoline, quinoxaline, pyrimidine, pyrazine, tetrazole, and pyrazole.
xe2x80x9cHeteroarylalkylxe2x80x9d means an alkyl containing a heteroaryl ring. For example: pyridinylmethyl, pyrimidinylethyl, and the like.
The material upon which the syntheses of the invention are performed are referred to as solid supports, beads, and resins. These terms are intended to include: beads, pellets, disks, fibers, gels, or particles such as cellulose beads, pore-glass beads, silica gels, polystyrene beads optionally cross-linked with divinylbenzene and optionally grafted with polyethylene glycol and optionally functionalized with amino, hydroxy, carboxy, or halo groups, grafted co-poly beads, polyacrylamide beads, latex beads, dimethylacrylamide beads optionally cross-linked with N,Nxe2x80x2-bis-acryloyl ethylene diamine, glass particles coated with hydrophobic polymer, etc., i.e., material having a rigid or semi-rigid surface; and soluble supports such as low molecular weight non-cross-linked polystyrene.
III. Optical Isomersxe2x80x94Diastereomersxe2x80x94Geometric Isomers
Some of the compounds described herein contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisometric forms which may be defined in terms of absolute stereochemistry as (R)- or (S)-, or as (D)- or (L)- for amino acids. The present invention is meant to include all such possible diastereomers as well as their racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended to include both (E)- and (Z)-geometric isomers. Likewise, all tautomeric forms are intended to be included.
IV. Assays for Determining Biological Activity
Materials
Plasmepsin II was obtained from Daniel E. Goldberg, Washington University. The plasmepsin II substrate, (DABCYL)-xcex3-aminobutyric acid-Glu-Arg-Met-Phe-Leu-Ser-Phe-Pro-EDANS, and the cathepsin D substrate, DABCYL-xcex3-aminobutyric acid-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-EDANS or Ac-Glu-Glu(EDANS)-Lys-Pro-Ile-Met-Phe-Phe-Arg-Leu-Gly-Lys-(DABCYL)-Glu-NH2 (Sergei V. Gulnik and John W. Erickson, National Cancer Institute) were purchased as a custom synthesis from AnaSpec, Inc., 2149 O""Toole Avenue, Suite F, San Jose, Calif. 95131.
Cathepsin D from human liver was purchased from ART Biochemicals, Athens Research Technology, PO Box 5494, Athens, Ga. 30604.
Method for Plasmepsin II
The assay mix contained 50 mM sodium acetate (pH 5.0), 1 mg/ml BSA, 0.01% Tween 20, 12.5% glycerol, 18% DMSO and 12 xcexcM plasmepsin substrate. Twenty five xcexcL of the assay mix was added to each well of the 96-well microtiter plate containing dried down bead eluate or empty control wells. The plates were then sonicated and mixed. Twenty five xcexcL of 8 nM plasmepsin II in 50 mM sodium acetate (pH 5.0), 1 mg/ml BSA, 0.01% Tween 20, and 12.5% glycerol, were added to the assay mix. The final concentrations were 4 nM plasmepsin II, 6 xcexcM plasmepsin substrate, 9% DMSO, 50 mM sodium acetate (pH 5.0), 1 mg/ml BSA, 0.01% Tween 20, and 12.5% glycerol. The reaction was incubated for 10 minutes at 25xc2x0 C. and then quenched by the addition of 25 xcexcL of 1 M Tris (pH 8.5) and 50% DMSO to achieve a final concentration of 0.33 M Tris and 23% DMSO. The EDANS fluorescence was measured using the Tecan, SLT FluoStar fluorescence plate reader with an excitation filter of 350 nm and an emission filter of 510 nm. The background was determined by 25 xcexcL of 50 mM sodium acetate (pH 5.0), 1 mg/ml BSA, 0.01% Tween 20, and 12.5% glycerol without enzyme.
Method for Cathepsin D
The assay mix contained 25 mM sodium formate (pH 3.5), 1 mg/ml BSA, 12% DMSO and 12 xcexcM cathepsin D substrate. Twenty five xcexcL of the assay mix were added to each well of the 96-well microtiter plate containing dried down bead eluate or empty control wells. The plates were then sonicated and mixed. Twenty five xcexcL of 1.6 nM cathepsin D in 25 mM sodium formate (pH 3.5), and 1 mg/ml BSA, were added to the assay mix. The final concentrations were 0.8 nM cathepsin D, 6 xcexcM cathepsin D substrate, 6% DMSO, 25 mM sodium formate (pH 3.5), and 1 mg/ml BSA. The reaction was incubated for 10 minutes at 25xc2x0 C. and then quenched by the addition of 25 xcexcL of 1 M Tris (pH 8.5) and 50% DMSO to achieve a final concentration of 0.33 M Tris and 21% DMSO. The EDANS fluorescence was measured as stated above. The background was determined by 25 xcexcL of 50 mM sodium formate (pH 3.5), and 1 mg/ml BSA without enzyme.
V. Methods of Synthesis
The compounds of the present invention may be prepared according to the following methods. In carrying out the syntheses, one typically begins with a quantity of solid support that will provide enough compound after cleavage from the solid support for biological testing in the herein described assays. In the case where the solid support is TentaGel(trademark), it is recommended that approximately 0.5 g of beads of about 180 microns in diameter, with a loading capacity of about 300 picoM per bead, be used. As the chemical yield of compounds after photolysis typically ranges from approximately 20% up to 60%, this quantity will provide a yield (approximately  greater than 10 mg) sufficient for biological testing in the given protease assays. For actual synthesis, the appropriate reagents and reaction conditions are applied to a reaction vessel containing the specified quantity of beads. During the syntheses, the beads may be washed free of any excess reagents or by-products before proceeding to the next reaction.
A. Scheme 1: Synthesis of Hydroxyphosphonates.
A batch of amino-functionalized PEG-grafted polystyrene beads such as TentaGel(trademark) 1 is used in the synthesis. The batch was first treated with bis-Fmoc lysine to increase the loading capacity of the resin. The Fmoc groups were removed using piperidine under standard conditions to which was then added 4-bromomethyl-3-nitrobenzoic acid 2. This was accomplished by the following method. The amine resin was suspended in DMF, and treated with a solution of 2, HOBt, DIC in DMF. The suspension was shaken overnight, then drained and the resin was washed with DCM. The resin 3 was dried overnight in vacuum.
Resin 3 was reacted with a unique amino-TBS ether to generate resin 4. The coupling of each amine occurred through displacement of the linker bromide and formation of a new carbon-nitrogen bond. Two cycles of reactions were performed to ensure complete conversion. In the first cycle, the amine was added to a suspension of resin 3 in THF and the mixture was shaken overnight. The mixture was drained and the resin was washed with THF. The THF solution containing the excess amine was then concentrated, taken up in DCM, washed with aqueous sodium bicarbonate, dried over sodium sulfate and concentrated. The residue was taken up in DMF and reacted with the same resin for the second reaction cycle. Lithium iodide was added to the suspension and the mixture was shaken overnight. The suspension was drained and the resin was washed with DMF, methanol, DCM and dried overnight in vacuum to give resin 4. After coupling, a small portion of each batch of resin was removed and titrated with picric acid to determine the extent of amine loading as a quality control for the reaction in this step.
The amine 4 was acylated by using acid chlorides. An acid chloride was added to a suspension of amine resin 4 in pyridine. The mixture was shaken overnight, drained and the resin was washed with DMF, methanol and DCM. When using acid chloride 6, the chloromethylacetoxy group was removed with hydrazine in methanol for 1 hour at r.t., drained, washed with DCM and acetonitrile. The resin 8 so obtained was shaken with an isocyanate in acetonitrile in the presence of a base overnight. The resin was finally drained and washed with DMF, methanol, DCM. This gave the carbamate derivatized resin 9.
Either resin 5 or 9 was converted to the corresponding aldehyde resin 10 by deprotection and oxidation. Resin 5 or 9 was treated with dilute hydrochloric acid in methanol for 5-8 hr to remove the t-butyldimethylsilyl (TBS) protecting group. The resin was then washed with DMF, methanol and DCM. The resulting alcohols were oxidized to the corresponding aldehydes by the following method: To a suspension of the resin in DMSO was added a solution of IBX in DMSO and the mixture was shaken overnight. The suspension was drained and the resin was washed with DMSO and treated with another solution of IBX in DMSO for 4 hr. The mixture was then drained and the resin washed with DMSO, methanol, DCM and dried overnight in vacuum to give the aldehydes 10.
To a suspension of the resin 10 in DCM was added a phosphite followed by triethylamine and the mixture was shaken overnight. The suspension was drained and the resin 11 was washed with DMF, methanol, DCM. Amides of Formula I (i.e., compounds 12) were cleaved from resin compounds 11 by exposing them to UV light (ca. 360 nM) for 15-180 minutes at 25-50xc2x0 C. in a suitable solvent such as methanol.
B. Scheme 2: Synthesis of Hydroxystatine Amides.
In this chemistry, the resin bound aldehyde 10 (from Scheme 1) was converted to the diacetate ester 15 by a Wittig reaction, followed by catalytic dihydroxylation of the alkene and protection of the diol as a diacetate. The resin bound aldehydes 10 suspended in THF were reacted with (t-butoxycarbonylmethylene)-triphenylphosphororane overnight. After washing with THF, methanol and DCM, the xcex1,xcex2-unsaturated esters 13 were suspended in acetone-water (1:1 mixture) and NMO was added along with a solution of osmium tetroxide in water. The mixture was shaken overnight, drained and the resin was washed with water, pyridine, DMF, methanol and DCM. Protection of the diol 14 was accomplished by treatment of the resin with a solution of acetic anhydride in pyridine containing a catalytic amount of DMAP for 18 hr. The resin was subsequently washed with DMF, methanol and DCM and dried overnight in vacuum to give the diacetate resin 15.
The ester-diacetate 15 was converted to the corresponding acid, which was then coupled with an amine. Deprotection of the diacetate amide resin 19 then led to the resin 20. Hydrolysis of ester 15 was accomplished by treatment with neat TFA for 2 hr. The resin was then washed with DMF, methanol and DCM, and suspended in a small amount of a 1:1 mixture of DMF:pyridine. Pentafluorophenyl trifluoroacetate was added along with pentafluorophenol and the mixture was shaken 1 hr at r.t., then drained. The resin bound activated ester 18 was washed briefly with DMF and treated overnight with a DMF solution of an amine. After washing with DMF, methanol and DCM, the amide-diacetate 19 was shaken 2 hr in a solution of hydrazine in methanol to afford resin bound diol 20. The resin bound diol 20 was washed with DMF, methanol and DCM and dried overnight in vacuum. Amides of Formula I (i.e., compounds 21) may be cleaved from resin compounds 20 by exposing them to UV light (ca. 360 nM) for 15-180 minutes at 25-50xc2x0 C. in a suitable solvent such as methanol.